Correspondence between SARSr-CoV ORF3ab-E gene sequences, highlighting the designed primer pair and probe-binding site. (a) Representative sequences of SARS-CoV-2 and SARSr-CoV were aligned to identify the highly conserved regions of the ORF3ab-E gene. The target regions of the designed primer pair and probe were highlighted. Matching residues are represented as dots. (b) Sequence tree reconstructed using the BEAST program [34] to visualize the detection scope of the designed universal ORF3ab-E gene detection primers. The colored sequences correspond to SARSr-CoV constituents that can be detected by the developed assay. The hosts of each constituent strain are shown using a circle (human host), diamond (civet), square (monkey), triangle (badger), star (bat), inverted triangle (pangolin), and spade (anteater). 2.2 Optimization and sensitivity evaluation of the PNA-mediated RT-qPCR assay for N gene-specific detection.